Immunostimulatory nucleic acid molecules

ABSTRACT

Nucleic acids containing unmethylated CpG dinucleotides and therapeutic utilities based on their ability to stimulate an immune response and to redirect a Th2 response to a Th1 response in a subject are disclosed. Methods for treating atopic diseases, including atopic dermatitis, are disclosed.

RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 08/738,652, filed Oct. 30, 1996, which is acontinuation-in-part of U.S. patent application Ser. No. 08/386,063,filed Feb. 7, 1995, now issued as U.S. Pat. No. 6,194,388, which is acontinuation-in-part of U.S. patent application Ser. No. 08/276,358,filed Jul. 15, 1994, now abandoned.

GOVERNMENT SUPPORT

The work resulting in this invention was supported in part by NationalInstitute of Health Grant No. R29-AR42556-01. The U.S. Government maytherefore be entitled to certain rights in the invention.

BACKGROUND OF THE INVENTION

DNA Binds To Cell Membranes And Is Internalized

In the 1970's, several investigators reported the binding of highmolecular weight DNA to cell membranes (Lerner, R. A., W. Meinke, and D.A. Goldstein. 1971. “Membrane-associated DNA in the cytoplasm of diploidhuman lymphocytes”. Proc. Natl. Acad. Sci. USA 68:1212; Agrawal, S. K.,R. W. Wagner, P. K. McAllister, and B. Rosenberg. 1975.“Cell-surface-associated nucleic acid in tumorigenic cells made visiblewith platinum-pyrimidine complexes by electron microscopy”. Proc. Natl.Acad. Sci. USA 72:928). In 1985, Bennett et al. presented the firstevidence that DNA binding to lymphocytes is similar to a ligand receptorinteraction: binding is saturable, competitive, and leads to DNAendocytosis and degradation into oligonucleotides (Bennett, R. M., G. T.Gabor, and M. M. Merritt. 1985. “DNA binding to human leukocytes.Evidence for a receptor-mediated association, internalization, anddegradation of DNA”. J. Clin. Invest. 76:2182). Like DNA,oligodeoxyribonucleotides (ODNs) are able to enter cells in a saturable,sequence independent, and temperature and energy dependent fashion(reviewed in Jaroszewski, J. W., and J. S. Cohen. 1991. “Cellular uptakeof antisense oligodeoxynucleotides”. Advanced Drug Delivery Reviews6:235; Akhtar, S., Y. Shoji, and R. L. Juliano. 1992. “Pharmaceuticalaspects of the biological stability and membrane transportcharacteristics of antisense oligonucleotides”. In: Gene Regulation:Biology of Antisense RNA and DNA. R. P. Erickson, and J. G. Izant, eds.Raven Press, Ltd. New York, pp. 133; and Zhao, Q., T. Waldschmidt, E.Fisher, C. J. Herrera, and A. M. Krieg., 1994. “Stage specificoligonucleotide uptake in murine bone marrow B cell precursors”. Blood,84:3660). No receptor for DNA or ODN uptake has yet been cloned, and itis not yet clear whether ODN binding and cell uptake occurs through thesame or a different mechanism from that of high molecular weight DNA.

Lymphocyte ODN uptake has been shown to be regulated by cell activation.Spleen cells stimulated with the B cell mitogen LPS had dramaticallyenhanced ODN uptake in the B cell population, while spleen cells treatedwith the T cell mitogen Con A showed enhanced ODN uptake by T but not Bcells (Krieg, A. M., F. Gmelig-Meyling, M. F. Gourley, W. J. Kisch, L.A. Chrisey, and A. D. Steinberg. 1991. “Uptake ofoligodeoxyribonucleotides by lymphoid cells is heterogeneous andinducible”. Antisense Research and Development 1:161).

Immune Effects Of Nucleic Acids

Several polynucleotides have been extensively evaluated as biologicalresponse modifiers. Perhaps the best example is poly (I,C) which is apotent inducer of IFN production as well as a macrophage activator andinducer of NK activity (Talmadge, J. E., J. Adams, H. Phillips, M.Collins, B. Lenz, M. Schneider, E. Schlick, R. Ruffmann, R. H. Wiltrout,and M. A. Chirigos. 1985. “Immunomodulatory effects in mice ofpolyinosinic-polycytidylic acid complexed with poly-L-lysine andcarboxymethylcellulose”. Cancer Res. 45:1058; Wiltrout, R. H., R. R.Salup, T. A. Twilley, and J. E. Talmadge. 1985. “Immunomodulation ofnatural killer activity by polyribonucleotides”. J. Biol. Resp. Mod.4:512; Krown, S. E. 1986. “Interferons and interferon inducers in cancertreatment”. Sem. Oncol. 13:207; and Ewel, C. H., S. J. Urba, W. C. Kopp,J. W. Smith II, R. G. Steis, J. L. Rossio, D. L. Longo, M. J. Jones, W.G. Alvord, C. M. Pinsky, J. M. Beveridge, K. L. McNitt, and S. P.Creekmore. 1992. “Polyinosinic-polycytidylic acid complexed withpoly-L-lysine and carboxymethylcellulose in combination withinterleukin-2 in patients with cancer: clinical and immunologicaleffects”. Canc. Res. 52:3005). It appears that this murine NK activationmay be due solely to induction of IFN-β secretion (Ishikawa, R., and C.A. Biron. 1993. “IFN induction and associated changes in splenicleukocyte distribution”. J. Immunol. 150:3713). This activation wasspecific for the ribose sugar since deoxyribose was ineffective. Itspotent in vitro antitumor activity led to several clinical trials usingpoly (I,C) complexed with poly-L-lysine and carboxymethylcellulose (toreduce degradation by RNAse) (Talmadge, J. E., et al., 1985. citedsupra; Wiltrout, R. H., et al., 1985. cited supra); Krown, S. E., 1986.cited supra); and Ewel, C. H., et al., 1992. cited supra).Unfortunately, toxic side effects have thus far prevented poly (I,C)from becoming a useful therapeutic agent.

Guanine ribonucleotides substituted at the C8 position with either abromine or a thiol group are B cell mitogens and may replace “B celldifferentiation factors” (Feldbush, T. L., and Z. K. Ballas. 1985.“Lymphokine-like activity of 8-mercaptoguanosine: induction of T and Bcell differentiation”. J. Immunol. 134:3204; and Goodman, M. G. 1986.“Mechanism of synergy between T cell signals and C8-substituted guaninenucleosides in humoral immunity: B lymphotropic cytokines induceresponsiveness to 8-mercaptoguanosine”. J. Immunol. 136:3335).8-mercaptoguanosine and 8-bromoguanosine also can substitute for thecytokine requirement for the generation of MHC restricted CTL (Feldbush,T. L., 1985. cited supra), augment murine NK activity (Koo, G. C., M. E.Jewell, C. L. Manyak, N. H. Sigal, and L. S. Wicker. 1988. “Activationof murine natural killer cells and macrophages by 8-bromoguanosine”. J.Immunol. 140:3249), and synergize with IL-2 in inducing murine LAKgeneration (Thompson, R. A., and Z. K. Ballas. 1990.“Lymphokine-activated killer (LAK) cells. V. 8-Mercaptoguanosine as anIL-2-sparing agent in LAK generation”. J. Immunol. 145:3524). The NK andLAK augmenting activities of these C8-substituted guanosines appear tobe due to their induction of IFN (Thompson, R. A., et al. 1990. citedsupra). Recently, a 5′ triphosphorylated thymidine produced by amycobacterium was found to be mitogenic for a subset of human γδ T cells(Constant, P., F. Davodeau, M. -A. Peyrat, Y. Poquet, G. Puzo, M.Bonneville, and J. -J. Foumie. 1994. “Stimulation of human γδ T cells bynonpeptidic mycobacterial ligands” Science 264:267). This reportindicated the possibility that the immune system may have evolved waysto preferentially respond to microbial nucleic acids.

Several observations suggest that certain DNA structures may also havethe potential to activate lymphocytes. For example, Bell et al. reportedthat nucleosomal protein-DNA complexes (but not naked DNA) in spleencell supernatants caused B cell proliferation and immunoglobulinsecretion (Bell, D. A., B. Morrison, and P. VandenBygaart. 1990.“Immunogenic DNA-related factors”. J. Clin. Invest. 85:1487). In othercases, naked DNA has been reported to have immune effects. For example,Messina et al. have recently reported that 260 to 800 bp fragments ofpoly (dG)●(dC) and poly (dG● dC) were mitogenic for B cells (Messina, J.P., G. S. Gilkeson, and D. S. Pisetsky. 1993. “The influence of DNAstructure on the in vitro stimulation of murine lymphocytes by naturaland synthetic polynucleotide antigens”. Cell. Immunol.147:148).Tokunaga, et al. have reported that dG●dC induces IFN-γ and NK activity(Tokunaga, S. Yamamoto, and K. Namba. 1988. “A synthetic single-strandedDNA, poly(dG,dC), induces interferon-α/β and -γ, augments natural killeractivity, and suppresses tumor growth” Jpn. J. Cancer Res. 79:682).Aside from such artificial homopolymer sequences, Pisetsky et al.reported that pure mammalian DNA has no detectable immune effects, butthat DNA from certain bacteria induces B cell activation andimmunoglobulin secretion (Messina, J. P., G. S. Gilkeson, and D. S.Pisetsky. 1991. “Stimulation of in vitro murine lymphocyte proliferationby bacterial DNA”. J. Immunol. 147:1759). Assuming that these data didnot result from some unusual contaminant, these studies suggested that aparticular structure or other characteristic of bacterial DNA renders itcapable of triggering B cell activation. Investigations of mycobacterialDNA sequences have demonstrated that ODN which contain certainpalindrome sequences can activate NK cells (Yamamoto, S., T. Yamamoto,T. Kataoka, E. Kuramoto, O. Yano, and T. Tokunaga. 1992. “Uniquepalindromic sequences in synthetic oligonucleotides are required toinduce INF and augment INF-mediated natural killer activity”. J.Immunol. 148:4072; Kuramoto, E., O. Yano, Y. Kimura, M. Baba, T. Makino,S. Yamamoto, T. Yamamoto, T. Kataoka, and T. Tokunaga. 1992.“Oligonucleotide sequences required for natural killer cell activation”.Jpn. J. Cancer Res. 83:1128).

Several phosphorothioate modified ODN have been reported to induce invitro or in vivo B cell stimulation (Tanaka, T., C. C. Chu, and W. E.Paul. 1992. “An antisense oligonucleotide complementary to a sequence inIγ2b increases γ2b germline transcripts, stimulates B cell DNAsynthesis, and inhibits immunoglobulin secretion”. J. Exp. Med. 175:597;Branda, R. F., A. L. Moore, L. Mathews, J. J. McCormack, and G. Zon.1993. “Immune stimulation by an antisense oligomer complementary to therev gene of HIV-1”. Biochem. Pharmacol. 45:2037; McIntyre, K. W., K.Lombard-Gillooly, J. R. Perez, C. Kunsch, U. M. Sarmiento, J. D.Larigan, K. T. Landreth, and R. Narayanan. 1993. “A sensephosphorothioate oligonucleotide directed to the initiation codon oftranscription factor NFκB T65 causes sequence-specific immunestimulation”. Antisense Res. Develop. 3:309; and Pisetsky, D. S., and C.F. Reich. 1993. “Stimulation of murine lymphocyte proliferation by aphosphorothioate oligonucleotide with antisense activity for herpessimplex virus”. Life Sciences 54:101). These reports do not suggest acommon structural motif or sequence element in these ODN that mightexplain their effects.

The CREB/ATF Family Of Transcription Factors And Their Role InReplication

The cAMP response element binding protein (CREB) and activatingtranscription factor (ATF) or CREB/ATF family of transcription factorsis a ubiquitously expressed class of transcription factors of which 11members have so far been cloned (reviewed in de Groot, R. P., and P.Sassone-Corsi: “Hormonal control of gene expression: Multiplicity andversatility of cyclic adenosine 3′,5′-monophosphate-responsive nuclearregulators”. Mol. Endocrin. 7:145, 1993; Lee, K. A. W., and N. Masson:“Transcriptional regulation by CREB and its relatives”. Biochim.Biophys. Acta 1174:221, 1993.). They all belong to the basicregion/leucine zipper (bZip) class of proteins. All cells appear toexpress one or more CREB/ATF proteins, but the members expressed and theregulation of mRNA splicing appear to be tissue-specific. Differentialsplicing of activation domains can determine whether a particularCREB/ATF protein will be a transcriptional inhibitor or activator. ManyCREB/ATF proteins activate viral transcription, but some splicingvariants which lack the activation domain are inhibitory. CREB/ATFproteins can bind DNA as homo- or hetero- dimers through the cAMPresponse element, the CRE, the consensus form of which is theunmethylated sequence TGACGTC (binding is abolished if the CpG ismethylated) (Iguchi-Ariga, S. M. M., and W. Schaffner: “CpG methylationof the cAMP-responsive enhancer/promoter sequence TGACGTCA abolishesspecific factor binding as well as transcriptional activation”. Genes &Develop. 3:612, 1989.).

The transcriptional activity of the CRE is increased during B cellactivation (Xie, H. T. C. Chiles, and T. L. Rothstein: “Induction ofCREB activity via the surface Ig receptor of B cells”. J. Immunol.151:880, 1993.). CREB/ATF proteins appear to regulate the expression ofmultiple genes through the CRE including immunologically important genessuch as fos, jun B, Rb-1, IL-6, IL-1 (Tsukada, J., K. Saito, W. R.Waterman, A. C. Webb, and P. E. Auron: “Transcription factors NF-IL6 andCREB recognize a common essential site in the human prointerleukin 1βgene”. Mol. Cell. Biol. 14:7285, 1994; Gray, G. D., O. M. Hernandez, D.Hebel, M. Root, J. M. Pow-Sang, and E. Wickstrom: “Antisense DNAinhibition of tumor growth induced by c-Ha-ras oncogene in nude mice”.Cancer Res. 53:577, 1993), IFN-β (Du, W., and T. Maniatis: “An ATF/CREBbinding site protein is required for virus induction of the humaninterferon B gene”. Proc. Natl. Acad. Sci. USA 89:2150, 1992), TGF-β1(Asiedu, C. K., L. Scott, R. K. Assoian, M. Ehrlich: “Binding ofAP-1/CREB proteins and of MDBP to contiguous sites downstream of thehuman TGF-B1 gene”. Biochim. Biophys. Acta 1219:55, 1994.), TGF-β2,class II MHC (Cox, P. M., and C. R. Goding: “An ATF/CREB binding motifis required for aberrant constitutive expression of the MHC class II DRapromoter and activation by SV40 T-antigen”. Nucl. Acids Res. 20:4881,1992.), E-selectin, GM-CSF, CD-8α, the germline Iga constant regiongene, the TCR Vβ gene, and the proliferating cell nuclear antigen(Huang, D., P. M. Shipman-Appasamy, D. J. Orten, S. H. Hinrichs, and M.B. Prystowsky: “Promoter activity of the proliferating-cell nuclearantigen gene is associated with inducible CRE-binding proteins ininterleukin 2-stimulated T lymphocytes”. Mol. Cell. Biol. 14:4233,1994.). In addition to activation through the cAMP pathway, CREB canalso mediate transcriptional responses to changes in intracellular Ca⁺⁺concentration (Sheng, M., G. McFadden, and M. E. Greenberg: “Membranedepolarization and calcium induce c-fos transcription viaphosphorylation of transcription factor CREB”. Neuron 4:571, 1990).

The role of protein-protein interactions in transcriptional activationby CREB/ATF proteins appears to be extremely important. There areseveral published studies reporting direct or indirect interactionsbetween NFKB proteins and CREB/ATF proteins (Whitley, et. al., (1994)Mol. & Cell. Biol. 14:6464; Cogswell, et al., (1994) J. Immun. 153:712;Hines, et al., (1993) Oncogene 8:3189; and Du, et al., (1993) Cell74:887. Activation of CREB through the cyclic AMP pathway requiresprotein kinase A (PKA), which phosphorylates CREB³⁴¹ on ser¹³³ andallows it to bind to a recently cloned protein, CBP (Kwok, R. P. S., J.R. Lundblad, J. C. Chrivia, J. P. Richards, H. P. Bachinger, R. G.Brennan, S. G. E. Roberts, M. R. Green, and R. H. Goodman: “Nuclearprotein CBP is a coactivator for the transcription factor CREB”. Nature370:223, 1994; Arias, J., A. S. Alberts, P. Brindle, F. X. Claret, T.Smea, M. Karin, J. Feramisco, and M. Montminy: “Activation of cAMP andmitogen responsive genes relies on a common nuclear factor”. Nature370:226, 1994.). CBP in turn interacts with the basal transcriptionfactor TFIIB causing increased transcription. CREB also has beenreported to interact with dTAFII 110, a TATA binding protein-associatedfactor whose binding may regulate transcription (Ferreri, K., G. Gill,and M. Montminy: “The cAMP-regulated transcription factor CREB interactswith a component of the TFIID complex”. Proc. Natl. Acad. Sci. USA91:1210, 1994.). In addition to these interactions, CREB/ATF proteinscan specifically bind multiple other nuclear factors (Hoeffler, J. P.,J. W. Lustbader, and C. -Y. Chen: “Identification of multiple nuclearfactors that interact with cyclic adenosine 3′,5′-monophosphate responseelement-binding protein and activating transcription factor-2 byprotein-protein interactions”. Mol. Endocrinol. 5:256, 1991) but thebiologic significance of most of these interactions is unknown. CREB isnormally thought to bind DNA either as a homodimer or as a heterodimerwith several other proteins. Surprisingly, CREB monomers constitutivelyactivate transcription (Krajewski, W., and K. A. W. Lee: “A monomericderivative of the cellular transcription factor CREB functions as aconstitutive activator”. Mol. Cell. Biol. 14:7204, 1994.).

Aside from their critical role in regulating cellular transcription, ithas recently been shown that CREB/ATF proteins are subverted by someinfectious viruses and retroviruses, which require them for viralreplication. For example, the cytomegalovirus immediate early promoter,one of the strongest known mammalian promoters, contains eleven copiesof the CRE which are essential for promoter function (Chang, Y. -N., S.Crawford, J. Stall, D. R. Rawlins, K. -T. Jeang, and G. S. Hayward: “Thepalindromic series I repeats in the simian cytomegalovirus majorimmediate-early promoter behave as both strong basal enhancers andcyclic AMP response elements”. J. Virol. 64:264, 1990). At least some ofthe transcriptional activating effects of the adenovirus E1A protein,which induces many promoters, are due to its binding to the DNA bindingdomain of the CREB/ATF protein, ATF-2, which mediates EIA inducibletranscription activation (Liu, F., and M. R. Green: “Promoter targetingby adenovirus E1a through interaction with different cellularDNA-binding domains”. Nature 368:520, 1994). It has also been suggestedthat E1A binds to the CREB-binding protein, CBP (Arany, Z., W. R.Sellers, D. M. Livingston, and R. Eckner: “E1A-associated p300 andCREB-associated CBP belong to a conserved family of coactivators”. Cell77:799, 1994). Human T lymphotropic virus-I (HTLV-1), the retroviruswhich causes human T cell leukemia and tropical spastic paresis, alsorequires CREB/ATF proteins for replication. In this case, the retrovirusproduces a protein, Tax, which binds to CREB/ATF proteins and redirectsthem from their normal cellular binding sites to different DNA sequences(flanked by G- and C-rich sequences) present within the HTLVtranscriptional enhancer (Paca-Uccaralertkun, S., L. -J. Zhao, N. Adya,J. V. Cross, B. R. Cullen, I. M. Boros, and C. -Z. Giam: “In vitroselection of DNA elements highly responsive to the human T-celllymphotropic virus type I transcriptional activator, Tax”. Mol. Cell.Biol. 14:456, 1994; Adya, N., L. -J. Zhao, W. Huang, I. Boros, and C.-Z. Giam: ”Expansion of CREB's DNA recognition specificity by Taxresults from interaction with Ala-Ala-Arg at positions 282-284 near theconserved DNA-binding domain of CREB”. Proc. Natl. Acad. Sci. USA91:5642, 1994).

SUMMARY OF THE INVENTION

The instant invention is based on the finding that certain nucleic acidscontaining unmethylated cytosine-guanine (CpG) dinucleotides activatelymphocytes in a subject and redirect a subject's immune response from aTh2 to a Th1 (e.g. by inducing monocytic cells and other cells toproduce Th1 cytokines, including IL-12, IFN-γ and GM-CSF). Based on thisfinding, the invention features, in one aspect, novel immunostimulatorynucleic acid compositions.

In a preferred embodiment, the immunostimulatory nucleic acid contains aconsensus mitogenic CpG motif represented by the formula: 5′ X1CGX2 3′

wherein X₁ is selected from the group consisting of A,G and T; and X₂ isC or T.

In a particularly preferred embodiment an immunostimulatory nucleic acidmolecule contains a consensus mitogenic CpG motif represented by theformula: 5′ X1X2CGX3X4 3′

wherein C and G are unmethylated; and X₁, X₂, X₃ and X₄ are nucleotides.

Enhanced immunostimulatory activity of human cells occurs where X₁X₂ isselected from the group consisting of GpT, GpG, GpA and ApA and/or X₃X₄is selected from the group consisting of TpT, CpT and GpT (Table 5). Forfacilitating uptake into cells, CpG containing immunostimulatory nucleicacid molecules are preferably in the range of 8 to 40 base pairs insize. However, nucleic acids of any size (even many kb long) areimmunostimulatory if sufficient immunostimulatory motifs are present,since such larger nucleic acids are degraded into oligonucleotidesinside of cells. Preferred synthetic oligonucleotides do not include aGCG trinucleotide sequence at or near the 5′ and/or 3′ terminals and/orthe consensus mitogenic CpG motif is not a palindrome. Prolongedimmunostimulation can be obtained using stabilized oligonucleotides,particularly phosphorothioate stabilized oligonucleotides.

In a second aspect, the invention features useful therapies, which arebased on the immunostimulatory activity of the nucleic acid molecules.For example, the immunostimulatory nucleic acid molecules can be used totreat, prevent or ameliorate an immune system deficiency (e.g., a tumoror cancer or a viral, fungal, bacterial or parasitic infection in asubject). In addition, immunostimulatory nucleic acid molecules can beadministered to stimulate a subject's response to a vaccine.

Further, by redirecting a subject's immune response from Th2 to Th1, theinstant claimed nucleic acid molecules can be administered to treat orprevent the symptoms of asthma. In addition, the instant claimed nucleicacid molecules can be administered in conjunction with a particularallergen to a subject as a type of desensitization therapy to treat orprevent the occurrence of an allergic reaction.

Further, the ability of immunostimulatory nucleic acid molecules toinduce leukemic cells to enter the cell cycle supports the use ofimmunostimulatory nucleic acid molecules in treating leukemia byincreasing the sensitivity of chronic leukemia cells and thenadministering conventional ablative chemotherapy, or combining theimmunostimulatory nucleic acid molecules with another immunotherapy.

Other features and advantages of the invention will become more apparentfrom the following detailed description and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-C are graphs plotting dose-dependent IL-6 production in responseto various DNA sequences in T cell depleted spleen cell cultures. A. E.coli DNA (●) and calf thymus DNA (▪) sequences and LPS (at 10× theconcentration of E. coli and calf thymus DNA) (♦). B. Controlphosphodiester oligodeoxynucleotide (ODN) ^(5′)ATGGAAGGTCCAGTGTTCTC^(3′)(SEQ ID NO: 1) (▪) and two phosphodiester CpG ODN^(5′)ATCGACCTACGTGCGTTCTC^(3′) (SEQ ID NO:2) (♦) and^(5′)TCCATAACGTTCCTGATGCT^(3′) (SEQ ID NO:3) (●). C. Controlphosphorothioate ODN ^(5′)GCTAGATGTTAGCGT^(3′) (SEQ ID NO:4) (▪) and twophosphorothioate CpG ODN ^(5′)GAGAACGTCGACCTTCGAT^(3′) (SEQ ID NO:5) (♦)and ^(5′)GCATGACGTTGAGCT^(3′) (SEQ ID NO:6) (●). Data present themean±standard deviation of triplicates.

FIG. 2 is a graph plotting IL-6 production induced by CpG DNA in vivo asdetermined 1-8 hrs after injection. Data represent the mean fromduplicate analyses of sera from two mice. BALB/c mice (two mice/group)were injected iv. with 100 μl of PBS (□) or 200 μg of CpGphosphorothioate ODN 5′ TCCATGACGTTCCTGATGCT 3′ (SEQ ID NO:7) (▪) ornon-CpG phosphorothioate ODN 5′ TCCATGAGCTTCCTGAGTCT 3′ (SEQ ID NO:8)(♦).

FIG. 3 is an autoradiograph showing IL-6 mRNA expression as determinedby reverse transcription polymerase chain reaction in liver, spleen, andthymus at various time periods after in vivo stimulation of BALB/c mice(two mice/group) injected iv with 100 μl of PBS, 200 μg of CpGphosphorothioate ODN 5′ TCCATGACGTTCCTGATGCT 3′ (SEQ ID NO:7) or non-CpGphosphorothioate ODN 5′ TCCATGAGCTTCCTGAGTCT 3′ (SEQ ID NO:8).

FIG. 4A is a graph plotting dose-dependent inhibition of CpG-induced IgMproduction by anti-L-6. Splenic B-cells from DBA/2 mice were stimulatedwith CpG ODN ^(5′)TCCAAGACGTTCCTGATGCT^(3′) (SEQ ID NO:9) in thepresence of the indicated concentrations of neutralizing anti-IL-6 (♦)or isotype control Ab (●) and IgM levels in culture supernatantsdetermined by ELISA. In the absence of CpG ODN, the anti-L-6 Ab had noeffect on IgM secretion (▪).

FIG. 4B is a graph plotting the stimulation index of CpG-induced splenicB cells cultured with anti-IL-6 and CpG S-ODN 5′ TCCATGACGTTCCTGATGCT 3′(SEQ ID NO:7) (♦) or anti- L-6 antibody only (▪). Data present themean±standard deviation of triplicates.

FIG. 5 is a bar graph plotting chloramphenicol acetyltransferase (CAT)activity in WEHI-231 cells transfected with a promoter-less CATconstruct (pCAT), positive control plasmid (RSV), or L-6 promoter-CATconstruct alone or cultured with CpG 5′ TCCATGACGTTCCTGATGCT 3′ (SEQ IDNO:7) or non-CpG 5′ TCCATGAGCTTCCTGAGTCT 3′ (SEQ ID NO:8)phosphorothioate ODN at the indicated concentrations. Data present themean of triplicates.

FIG. 6 is a schematic overview of the immune effects of theimmunostimulatory unmethylated CpG containing nucleic acids, which candirectly activate both B cells and monocytic cells (includingmacrophages and dendritic cells) as shown. The immunostimulatoryoligonucleotides do not directly activate purified NK cells, but renderthem competent to respond to IL-12 with a marked increase in their IFN-γ

1-18. (Canceled)
 19. A method for preventing or suppressingantigen-stimulated, eosinophilic inflammation in an antigen-exposedsubject comprising administering to the subject an isolatedimmunostimulatory oligonucleotide comprising X1X2CGX3X4, wherein C and Gare unmethylated and X1, X2, X3 and X4 are nucleotides and wherein theimmunostimulatory oligonucleotide is between 6 and 100 bases in length,in an amount to suppress a Th2 immune response, whereby eosinophilicinflammation is prevented or suppressed.
 20. The method of claim 19,wherein the immunostimulatory oligonucleotide includes a nucleotidesequence consisting of 5′-purine-purine-CG-pyrimidine-pyrimidine-3′. 21.The method of claim 20, wherein the nucleotide sequence consists ofAACGTT.
 22. The method of claim 19, wherein the immunostimulatoryoligonucleotide comprises a nucleotide sequence selected from the groupconsisting of GTCGTT, GTCGCT, GTCGGT, GGCGTT, GGCGCT, GGCGGT, GACGTT,GACGCT, GACGGT, AACGTT, AACGCT and AACGGT.
 23. The method of claim 19,wherein the subject has asthma, allergic rhinitis, eczema, hay fever orurticaria.
 24. The method of claim 19, wherein the eosinophilicinflammation occurs in a tissue affected by asthma, allergic rhinitis,eczema, hay fever or urticaria.
 25. The method of claim 19, wherein theeosinophilic inflammation is in the lung.
 26. The method of claim 25,wherein the subject has asthma.
 27. The method of claim 19, wherein theimmunostimulatory oligonucleotide is 8-100 bases in length.
 28. Themethod of claim 19, wherein the immunostimulatory oligonucleotide is8-40 bases in length.
 29. A method for boosting an immune response of asubject comprising administering to the subject an isolatedimmunostimulatory oligonucleotide comprising X1X2CGX3X4, wherein C and Gare unmethylated and X1, X2, X3 and X4 are nucleotides and wherein theimmunostimulatory oligonucleotide is between 6 and 100 bases in length,and wherein an increase in activation of the subject's lymphocytes or NKcells indicates that the subject's immune response has been boosted. 30.The method of claim 29, wherein the activation of the subject'slymphocytes or NK cells is lymphocyte proliferation.
 31. The method ofclaim 29, wherein the activation of the subject's lymphocytes or NKcells is IgM secretion.
 32. The method of claim 29, wherein theactivation of the subject's lymphocytes or NK cells is increasedexpression of IL-12 and IFN-gamma.
 33. The method of claim 29, whereinthe subject has an immune system deficiency.
 34. The method of claim 33,wherein the immune system deficiency is an infection.
 35. The method ofclaim 34, wherein the infection is a bacterial, viral, fungal orparasitic infection.
 36. The method of claim 33, wherein the immunesystem deficiency is a bacterial infection by bacteria having bacterialantigens and wherein the increase in lymphocyte or NK activation isactivated B cell with antigen receptors specific for the bacterialantigens.
 37. The method of claim 33, wherein the immune systemdeficiency is cancer.
 38. The method of claim 30, wherein theimmunostimulatory oligonucleotide is 8-100 bases in length.
 39. Themethod of claim 30, wherein the immunostimulatory oligonucleotide is8-40 bases in length.
 40. The method of claim 29, wherein theimmunostimulatory oligonucleotide is administered in conjunction with avaccine.
 41. The method of claim 29, wherein the immunostimulatoryoligonucleotide is not administered in conjunction with a vaccine. 42.The method of claim 29, wherein the immunostimulatory oligonucleotideincludes a nucleotide sequence consisting of5′-purine-purine-CG-pyrimidine-pyrimidine-3′.
 43. The method of claim42, wherein the nucleotide sequence consists of AACGTT.
 44. The methodof claim 29, wherein the immunostimulatory oligonucleotide comprises anucleotide sequence selected from the group consisting of GTCGTT,GTCGCT, GTCGGT, GGCGTT, GGCGCT, GGCGGT, GACGTT, GACGCT, GACGGT, AACGTT,AACGCT and AACGGT.
 45. The method of claim 29, wherein the subject hasasthma, allergic rhinitis, eczema, hay fever or urticaria.
 46. Themethod of claim 29, wherein the immune response occurs in a tissueaffected by eczema, allergic rhinitis, hay fever or urticaria.
 47. Themethod of claim 29, wherein the immune response occurs in the lung. 48.The method of claim 45, wherein the subject has asthma and the subjectdevelops a Th1 immune response to an allergen.
 49. The method of claim29, wherein the subject has a viral or a parasitic infection and theimmune response to the infection is boosted.
 50. The method of claim 49,wherein the infection is a viral infection.
 51. A method for shiftingthe immune response of a subject to an antigen toward a Th1 immuneresponse comprising administering to the subject an isolatedimmunostimulatory oligonucleotide comprising X1X2CGX3X4, wherein C and Gare unmethylated and X1, X2, X3 and X4 are nucleotides and wherein theimmunostimulatory oligonucleotide is between 6 and 100 bases in length,wherein detection of a Th1 type immune response by the subject indicatesthat the shift to the Th1 immune response has been achieved.
 52. Themethod of claim 51, wherein the shift to the Th1 immune response isfurther associated with suppression of a Th2 immune response.
 53. Themethod of claim 51, wherein the immunostimulatory oligonucleotideincludes a nucleotide sequence consisting of5′-purine-purine-CG-pyrimidine-pyrimidine-3′.
 54. The method of claim53, wherein the nucleotide sequence consists of AACGTT.
 55. The methodof claim 51, wherein the immunostimulatory oligonucleotide comprises anucleotide sequence selected from the group consisting of GTCGTT,GTCGCT, GTCGGT, GGCGTT, GGCGCT, GGCGGT, GACGTT, GACGCT, GACGGT, AACGTT,AACGCT and AACGGT.
 56. The method of claim 51, wherein the subject hasasthma, allergic rhinitis, eczema, hay fever or urticaria, and the shiftto the Th1 immune response prevents or suppresses eosinophilicinflammation in the subject.
 57. The method of claim 56, wherein theeosinophilic inflammation is in a tissue affected by asthma, allergicrhinitis, eczema, hay fever or urticaria
 58. The method of claim 56,wherein the eosinophilic inflammation is in the lung.
 59. The method ofclaim 51, wherein the subject has asthma and the shift to the Th1 immuneresponse prevents or suppresses eosinophil infiltration into the lung ofthe subject.
 60. The method of claim 51, wherein the subject has a viralor parasitic infection and the shift to the Th1 immune response booststhe immune response to the infection.
 61. The method of claim 60,wherein the infection is a viral infection.
 62. The method of claim 19,wherein the desired result is measured by detecting in a samplecontaining lymphocyte obtained from the immunostimulatoryoligonucleotide treated subject: (1) a lower level of IL-4 in theimmunostimulatory oligonucleotide treated subject as compared to anantigen-challenged control; or (2) a higher level of IL-12-and/or IFNgamma in the immunostimulatory oligonucleotide treated subject ascompared to an antigen-challenged control.
 63. The method of claim 19,wherein prevention or suppression of eosinophilic inflammation ismeasured by detecting lower levels of eosinophils in an inflammatoryinfiltrate in the lung in an immunostimulatory oligonucleotide treatedsubject as compared to an antigen-challenged control.
 64. The method ofclaim 51, wherein the immunostimulatory oligonucleotide is 8-100 basesin length.
 65. The method of claim 51, wherein the immunostimulatoryoligonucleotide is 8-40 bases in length.
 66. The method of claim 51,wherein the immunostimulatory oligonucleotide is administered inconjunction with a vaccine.
 67. The method of claim 51, wherein theimmunostimulatory oligonucleotide is not administered in conjunctionwith a vaccine.
 68. A method for preventing or reducingantigen-stimulated, granulocyte-mediated inflammation in a tissue of anantigen-sensitized subject comprising administering an isolatedimmunostimulatory oligonucleotide to the subject, wherein a reductionin, or the absence of, a Th2 type immune response measured in thesubject, or a reduction in, or the absence of, other clinical signs ofinflammation in the subject after antigen challenge, indicates that thedesired prevention or reduction in granulocyte-mediated inflammation hasbeen achieved.
 69. The method of claim 68, wherein the immunostimulatoryoligonucleotide includes a hexameric nucleotide sequence consisting of5′-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3′.
 70. The method ofclaim 69, wherein the hexameric nucleotide sequence consists of AACGTT.71. The method of claim 68, wherein the immunostimulatoryoligonucleotide comprises a hexameric nucleotide sequence selected fromthe group consisting of GTCGTT, GTCGCT, GTCGGT, GGCGTT, GGCGCT, GGCGGT,GACGTT, GACGCT, GACGGT, AACGTT, AACGCT and AACGGT.
 72. The method ofclaim 68, wherein the subject is suffering from an condition induced bythe sensitizing antigen selected from the group of inflammatoryconditions consisting of asthma, allergic rhinitis, atopic dermatitis,allergic conjunctivitis and cutaneous basophil hypersensitivity.
 73. Themethod of claim 68, wherein the inflammation is in skin or mucosa. 74.The method of claim 73, wherein the inflammation is in a respiratorytissue.
 75. The method of claim 68, wherein the subject is sufferingfrom asthma.
 76. The method of claim 68, wherein the desired result ismeasured by detecting in a sample containing lymphocytes obtained fromthe immunostimulatory oligonucleotide treated subject: (1) a lower levelof IL-4 in the immunostimulatory oligonucleotide treated subject ascompared to an antigen-challenged control; or (2) a higher level ofIL-12 and/or IFN gamma in the immunostimulatory oligonucleotide treatedsubject as compared to an antigen-challenged control.
 77. A method forboosting the immune responsiveness of a subject to a sensitizing antigenwithout immunization of the subject by the sensitizing antigencomprising administering an isolated immunostimulatory oligonucleotideto the subject, wherein an increase in the magnitude of the subject'simmune response to the sensitizing antigen indicates that the desiredboost to the subject's immune responsiveness has been achieved.
 78. Themethod of claim 77, wherein the immunostimulatory oligonucleotideincludes a hexameric nucleotide sequence consisting of5′-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3′.
 79. The method ofclaim 78, wherein the hexameric nucleotide sequence consists of AACGTT.80. The method of claim 77, wherein the immunostimulatoryoligonucleotide includes a hexameric nucleotide sequence is selectedfrom the group of sequences consisting of GTCGTT, GTCGCT, GTCGGT,GGCGTT, GGCGCT, GGCGGT, GACGTT, GACGCT, GACGGT, AACGTT, AACGCT andAACGGT.
 81. The method of claim 77, wherein the subject is sufferingfrom an inflammatory condition induced by the sensitizing antigenselected from the group of inflammatory conditions consisting of asthma,allergic rhinitis, atopic dermatitis, allergic conjunctivitis andcutaneous basophil hypersensitivity.
 82. The method of claim 81, whereinthe immune response is in skin or mucosa.
 83. The method of claim 82,wherein the immune response is in respiratory tissue.
 84. The method ofclaim 83, wherein the subject is suffering from asthma and the subject'simmune responsiveness to a respiratory allergen is boosted.
 85. Themethod of claim 77, wherein the antigen is presented by a pathogen andthe subject's immune responsiveness to an intracellular infection by thepathogen is boosted.
 86. The method of claim 85, wherein the pathogen isa virus.
 87. A method for shifting the immune response of a subject to asensitizing antigen toward a Th1 phenotype comprising administering anisolated immunostimulatory oligonucleotide to the subject, whereindetection of a Th1 type immune response by the subject indicates thatthe desired shift to the Th1 phenotype has been achieved.
 88. The methodof claim 87, wherein the immunostimulatory oligonucleotide includes ahexameric nucleotide sequence consisting of5′-Purine-Purine-[C]-[G]-Pyrimidine-Pyrimidine-3′.
 89. The method ofclaim 88, wherein the hexameric nucleotide sequence consists of AACGTT.90. The method of claim 87, wherein the immunostimulatoryoligonucleotide includes a hexameric nucleotide sequence is selectedfrom the group consisting of GTCGTT, GTCGCT, GTCGGT, GGCGTT, GGCGCT,GGCGGT, GACGTT, GACGCT, GACGGT, AACGTT, AACGCT and AACGGT.
 91. Themethod of claim 87, wherein the subject is suffering from aninflammatory condition induced by the sensitizing antigen selected fromthe group of inflammatory conditions consisting of asthma, allergicrhinitis, atopic dermatitis, allergic conjunctivitis and cutaneousbasophil hypersensitivity, and the shift to the Th1 phenotype reducesgranulocyte-mediated inflammation in the affected tissue.
 92. The methodof claim 91, wherein the affected tissue is skin or mucosa.
 93. Themethod of claim 92, wherein the affected tissue is respiratory tissue.94. The method of claim 93, wherein the subject is suffering from asthmaand the shift to the Th1 phenotype reduces eosinophil infiltration ofthe lung.
 95. The method of claim 87, wherein the subject is sufferingfrom an intracellular infection by a pathogen and the shift to the Th1phenotype strengthens the subject's immune response to the pathogen. 96.The method of claim 91, wherein the pathogen is a virus.
 97. The methodof claim 87, wherein the desired result is measured by detecting in asample containing lymphocytes obtained from the immunostimulatoryoligonucleotide treated subject: (1) a lower level of IL-4 in theimmunostimulatory oligonucleotide treated subject as compared to anantigen-challenged control; or (2) a higher level of IL-12 and/or IFNgamma in the immunostimulatory oligonucleotide treated subject ascompared to an antigen-challenged control.
 98. The method of claim 68,wherein reduction or suppression of inflammation is measured by assayinginflammatory infiltrate from the subject for a reduction in granulocytecounts in inflammatory infiltrate of an affected subject tissue asmeasured in an antigen challenged subject before and after ISS-ODNadministration or detection of lower levels of granulocyte counts in anISS-ODN treated subject as compared to an antigen-challenged control 99.The method of claim 48, wherein the allergen is pollen, animal dander ordust.